Tocotrienols: Exciting Biological and Pharmacological Properties of Tocotrienols and Naturally Occurring Compounds, Part II

δ-Tocotrienol plus AHA Step-1 diet in hypercholesterolemic subjects caused reductions in lipid parameters (14% to 18%) with 250 mg/d dose, and 500 mg/d resulted induction in these parameters. Although, α-tocopherol is the most bioavailable form of vitamin E. There are few reports on bioavailability of tocotrienols in humans. Pharmacokinetics and bioavailability of δ-tocotrienol was quantified on plasma levels of tocol isomers, cytokines, and microRNAs. Subjects were fed doses of 125 mg/d to 500 mg/d. Plasma samples collected between 0 h to 10 h, levels of tocols estimated by HPLC, which resulted dose-dependent increases in AUC0-10, Cmax0-∞, Tmaxh, t1/2h, Cl-T 1/h, Vd/f, keh-1. Maximum plasma levels of δ-tocotrienol were at 3 h (125 mg/d to 250 mg/d), 6 h (500 mg/d). Effects of 32 compounds were evaluated on TNF-α secretion, nitric oxide production, and gene expression (TNF-α, IL-1β, IL-6, iNOS activity) in PPAR-α knockout mice. Anticancer activities of thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, amiloride, quinine sulfate showed significant anti-proliferative properties in Hela cells, pancreatic, prostate, breast, lungs, melanoma, B-lymphocytes, T-cells (40% to 95%). Results of plasma total mRNAs after δ-tocotrienol feeding to hepatitis C patients revealed significant down-regulated gene expression of pro-inflammatory cytokines. A mixture of δ-tocotrienol, resveratrol, vitamin D3 (NS-3) were given two capsules/d or cellulose/olive oil as placebo to individuals with T2DM (24-weeks). Significant down-regulation (15% to 74%) of gene expression in diabetes biomarkers and decreases i n serum levels of fasting-glucose, HbA1c, hs-CRP, fasting-insulin, HOMA-IR, MDA (9% to 23%) were observed with NS-3 treated T2DM. Pure plasma mRNAs and miRNAs of pre-dose vs. post-dose of NS-3 treated samples were analyzed by Next Generation Sequencing (NGS). Venn diagrams have established genetic regulatory network images and canonical signaling pathways for mRNA, miRNA, and paired mRNA-miRNA.


Introduction
My journey of studying tocotrienols has started thirty years ago, when I reported the isolation and biological function of α-tocotrienol as hypocholestrolemic agent from barley first time in 1986 as reported in part I [1,2]. This was acknowledged by late Byron J Richards and Dr. Barry Tan in their articles [3][4][5]. In part II, the remaining published results of papers 10 to 17 are summarized in vitro and in vivo studies on the impact of various isomers of tocotrienols ( Figure 1) and other natural products on inflammation, cardiovascular, cancer, hepatitis C disease, Type 2 Diabetes (T2DM) and pharmacokinetics using several cell lines, experimental animal models and human subjects from 2011 to present day. Most of the information described here, are based on our published papers during last decade (2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020)(2021). All human studies (6 out of 8 papers in this article) were double-blind, Randomized, placebo-Controlled Trial (RCT). A non-probability convenience sampling technique was used. The protocol of each human study was registered with WHO regional office in Asia (World Health Organization Sri Lanka Clinical Trial Registry, Sri Lanka Center; srilanactr@gmail.com), after ethical approval by the Institutional Review Board of Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan. The registry number and date has been reported in each human study paper. The studies were carried out according to the guidelines provided by the United States Food and Drug Administration (FDA, 2003) at (AFIP), Rawalpindi, and National University of Medical Sciences, Rawalpindi, Pakistan. All participants of human studies have signed an informed consent form before start of the study. All papers were published in refereed journals.
It is well known that α-tocopherol is the most bioavailable form of vitamin E, but several animal and clinical studies have also demonstrated tocotrienols bioavailability to various tissues. It was also reported that the bio-discrimination of α-tocopherol (vitamin E) influences the rate of tocotrienol absorption, mainly due to high affinity of α-tocopherol with "α-Tocopherol Transfer Protein" (α-TTP), which mediates secretion of α-Tocopherol (100%) from the liver into the circulatory system and is much higher than α-tocotrienol (12%) or other tocotrienols [10,11]. There are few reports on bioavailability of tocotrienols in humans. Most studies were carried out with mixtures of tocotrienols + tocopherols rather than pure tocotrienols. Moreover, dietary α-tocopherol interferes with the bioavailability of tocotrienols and prevents absorption and delivery to organs and tissues [12,13]. Recently, Pharmacokinetics and bioavailability of Annatto-based δ-tocotrienol on plasma levels of α-, β-, γ-, δ-tocotrienol and tocopherols were quantified and in addition, several cytokines and microRNAs were also reported [14]. An open-label, randomized study was reported the pharmacokinetics and bioavailability of δ-tocotrienol in 33 healthy-fed subjects. In which, all subjects (11/dose) were randomly assigned to doses of 125, 250, or 500 mg/d. Plasma samples collected at 0, 1, 2, 3, 4, 6, 8, 10 h intervals and tocols (tocotrienols and tocopherols) were estimated by HPLC [14]. The results reported the effects of δ-tocotrienol on pharmacokinetic parameters of all eight isomers of tocol. Supplementation of 125, 250 and 500 mg/d doses of Annatto δ-tocotrienol have resulted in dose-dependent increases of (a) area under concentration-time curve (AUCt 0 -t 10 (Tables 2A-2D) [14]. Maximum plasma levels of δ-tocotrienol were observed at 3 h with doses of 125 and 250 mg/d, and 6 h with 500 mg/d. γ-Tocotrienol, β-tocotrienol, αtocotrienol, and δ-tocopherol, γ-tocopheol, β-tocopherol, α-tocopherol were appeared in the plasma after 2 h (Tables 2A-2D) [14]. Moreover, δ-tocotrienol treatment resulted in downregulation of eight cytokines and up-regulation of adiponectin, TGF-β1, and leptin ( Table  2). The gene expression of miR-34a (increased in bipolar disorder) was down-regulated, but expression of miR-107, miR-122a, and miR-132 (decreased in Alzheimer's disease) was up-regulated by δ-tocotrienol treatment (Table 3) [14]. These were the first results, which have described the effect of δ-tocotrienol on pharmacokinetics and bioavailability of all eight isomers of tocol. When tocotrienols are supplemented in absence of tocopherols, δ-tocotrienol has better bioavailability and δ-tocotrienol is converted stepwise to other tocotrienols/tocopherols as shown in Figure 4 [14]. These results have supported that tocotrienol, particularly δ-tocotrienol, as a dietary supplement might be useful in the prevention of age-related and chronic ailments [14]. Tocotrienols lowered serum lipid parameters below 500 mg/d while increased lipid parameters at higher dose of 750 mg/d compared to 250 mg/d [9]. These results were further supported by our earlier findings of inhibition of chymotrypsin-like activity of 20S rabbit muscle proteasome with 40 μM of δ-tocotrienols and activation with 80 μM [15]. Thus δ-tocotrienol has a novel property of concentration-dependent inhibition and activation. Recently, the bioavailability of various doses of δ-tocotrienol in healthy fed participants plasma has been reported, which showed dose-dependent increases in Area Under the Curve (AUC), maximum Concentration (C max ), and time to achieve maximum peak (T max ) which varies between 3 h to 4 h for isomers of tocotrienols and 3 h to 6 h for isomers of tocopherols at 125, 250, 500 mg doses [14]. The results were also reported about the safety and impact of δ-tocotrienols after administering higher doses (750 mg/d and 1000 mg/d) to healthy subjects on various pharmacokinetic parameters [16]. All subjects (3/dose) were randomly assigned to one of each dose 750 mg/d or 1000 mg/d. Blood samples were collected, and tocols (tocopherols and tocotrienols) were quantified by HPLC of plasma collected at 0, 1, 2, 4, 6, 8 h intervals [16]. The plasma samples of doses 750 mg and 1000 mg resulted in the elution of all isomers of (α-, β-, γ-, δ-) tocotrienols and tocopherols for each time intervals (0 h to 8 h). The tocotrienols (ng/ml) present in 750 mg dose were β-tocotrienol (7838) > γ-tocotrienol (5055) > δ-tocotrienol (4045) α-tocotrienol (1389) (Table 4A). Whereas, for tocopherols were δ-tocopherol (13117) > γ-tocopherol (5544) > (β-tocopherol (3269) α-Tocopherol (1389) (Table 4A). Similar results were obtained with 1000 mg/d of δ-tocotrienol treatment (Table 4B) [16].
The consumption of 750 and 1000 mg/d of tocotrienols resulted in dose-dependent increases of plasma AUCt 0 -t 8 (Table 5A, 5B) [16]. Similar results of these parameters were reported for δ-tocopherol, γ-tocopherol, (β-tocopherol except T max for α-Tocopherol was 6h [16]. These results indicated pharmacokinetics of higher doses of 750 mg and 1000 mg of δ-tocotrienol and confirmed that T max was 3 h to 4 h for all isomers tocol except α-Tocopherol (6 h). These higher doses of tocotrienols were found to be safe and might be useful for the treatments of various types of cancer, diabetes, and Alzheimer's disease [16]. Inflammation has been implicated in cancer, diabetes and cardiovascular disease [17][18][19]. The important role played by lipopolysaccharides (LPS) in up-regulating inflammation is well-established [20]. LPS is expressed on the outer membrane of gram-negative bacteria, and induces several pro-inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), IL-6, IL-8 and production of nitric oxide [20]. The 32 compounds of different categories of organic chemistry as shown in Table 6 were selected to find out potent inflammatory biomarkers. The Peroxisome Proliferator-Activated Receptor-α (PPAR-α) knockout female mice were selected for the study due to their different effects in LPS-induced macrophages of δ-tocotrienol, riboflavin, quercetin on secretion of TNF-α (activation) compared to corresponding wild type (C57BL/6) control (inhibition) group [21], and also due to the prolonged response to inflammatory stimuli [22]. Moreover, the PPARs mice contain nuclear receptors, which bind to fatty acid-derived ligands and activate the transcription of genes that govern lipid metabolism. The primary sites of activation of PPAR-α, which recognizes monounsaturated and polyunsaturated fatty acids and eicosanoids, are present in liver, heart, muscle, and kidney [23]. According to its role in regulating fatty acid metabolism, PPAR-α activates gene expression involved in fatty acid uptake (fatty acid binding protein), β-oxidation (medium chain acyl-CoA dehydrogenase, carnitine palmitoyl transferase I, and acyl-CoA oxidase), transport into peroxisomes (ATP-binding cassette transporters D2 and D3), and omega-oxidation of unsaturated fatty acids (cytochrome P-450, 4A1 and 4A3) [23]. Moreover, PPAR-α knockout mice also induce fatty acid catabolism and prevent hypertriglyceridemia, and its activation decreases glucose uptake, and causes a shift glucose use to fatty acid oxidation in cardiac muscle. Therefore, selective PPAR-α agonists that increase fatty acid catabolism without using lipid accumulation in the heart might be effective treatment for dyslipidemia [23]. The hypothesis was that compounds with those anti-inflammatory properties will be useful for treatment of diabetes, cardiovascular disease, and other diseases based on inflammation [23].
Cancer is second most common cause of death in the United State. There are over 100 different types of cancer associated with different human organs, predominantly breast, liver, pancreas, prostate, colon, rectum, lung, and stomach. The properties of pro-inflammatory (for treatment of various types of cancers), and anti-inflammatory (for cardiovascular disease and diabetes) compounds have been reported [17,18]. The major problem associated with development of anticancer drugs is their lack of solubility in aqueous solutions and severe side effects in cancer patients. Therefore, the anticancer properties, anti-proliferative, and pro-apoptotic activity of novel naturally occurring, or FDA approved, nontoxic, proteasome inhibitors/activators, mostly aqueous soluble ( Figure 5) were reported in cancer cell lines obtained from various organs [24]. The results of treatments of several compounds in cancer cell lines were found to be very effective in inducing apoptosis of cancer cells. Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, amiloride, and quinine sulfate have significant anti-proliferation properties in Hela cells (44% to 87%) with doses of 2.5 μM to 20 μM, compared to respective controls (Table 7 and Figures 8(1-4) [24]. However, thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, and quinine sulphate were effective in pancreatic, prostate, breast, lungs, melanoma, Blymphocytes, and T-cells (Jurkat: 40% to 95%) compared to respective controls (Table  7). In lung cancer cells, these compounds were effective between 5 μM to 40 μM (Table  7) [24]. The results of thiostrepton, 2-methoxyestradiol, δ-tocotrienol, and quercetin were very effective and induced apoptosis in the range of (70% to 92%) in Hela and liver cells. All these results were translated into possible IC 50 values of anticancer activities and IC 50 values of anti-proliferation properties of thiostrepton in most of these cell lines were between doses of 2.5 μM to 5 μM, dexamethasone 2.5 μM to 20 μM, lactone 40 μM to 80 M (Table 8) [24]. These results have demonstrated effectiveness of several naturaloccurring compounds with anti-proliferative properties against cancer cells of several organs of humans. Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol and quercetin are very effective for apoptosis of cancer cells in liver, pancreas, prostate, breast, lung, melanoma, B-lymphocytes, and T-cells. The results have provided an opportunity to test these compounds either individually or in combination as dietary supplements in humans for treatment of various types of cancers [24]. As mentioned earlier that δ-tocotrienol is a naturally occurring proteasome inhibitor, which has the capacity to inhibit proliferation and induce apoptosis in several cancer cells obtained from several organs of humans, and other cancer cell lines [24]. Moreover, results of plasma total mRNAs after δ-tocotrienol feeding to hepatitis C patients revealed significant inhibition in the expression of pro-inflammatory cytokines (TNF-α, VCAM1, proteasome subunits) and induction in the expression of ICAM1 and IFN-γ after post-treatment [25]. This down-regulation of proteasome subunits leads to autophagy, apoptosis of immune cells and several genes. The results reported RNA-sequence analysis of plasma total mRNAs obtained from δ-tocotrienol treatment of hepatitis C patients of pre-dose vs. post-dose on gene expression regulated by proteasome [25]. The data based on >1 and 8-fold expression changes of 2136 genes were fed into "Ingenuity Pathway Analyses (IPA)" for core analysis, which describes possible canonical pathways, upstream regulators diseases and functional metabolic networks [25]. The IPA of "molecules" indicated fold change in gene expression of 953 molecules, which covered several categories of biological biomarkers. Out of these, gene expression of 220 related to this study, 12 were up-regulated, and 208 downregulated after δ-tocotrienol treatment (Table 9A, 9B). The gene expression of transcription regulators (ceramide synthase 3 and Mohawk homeobox) was up-regulated, and gene expression of 208 molecules was down-regulated, involved in several biological functions (HSP90AB1, PSMC3, CYB5R4, NDUFB1, CYP2R1, TNFRF1B, VEGFA, GPR65, PIAS1, SFPQ, GPS2, EIF3F, GTPBP8, EIF4A1, HSPA14, TLR8, TUSSC2) [25]. IPA of "causal network" indicated gene regulators (676), in which 76 down-regulated (26s proteasomes, interleukin cytokines, and PPAR-ligand-PPA-Retinoic acid-RXRα, PPARγ-ligand-PPARγ-Retinoic acid-RARα, IL-21, IL-23) with significant P-values (Table 9B) [25]. The IPA of "diseases and functions" regulators (85) were involved with cAMP, STAT2, 26S proteasome, CSF1, IFNγ, LDL, TGFA, and microRNA-155-5p, miR-223, miR-21-5p, and "upstream analysis" (934) showed 57 up-regulated (mainly 38 microRNAs) and 64 gene regulators were down-regulated (IL-2, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-24, IL-27, IL-32), interferon β-1α, interferon γ, TNF-α, STAT2, NOX1, prostaglandin J2, NF-κB, IκB, TCF3, and also miRNA-15, miRNA-124, miRNA-218-5P with significant activation of Z-Score (P<0.05) [25]. The effect of δ-tocotrienol treatment to hepatitis C on "canonical pathways (360)" also described of only 33 in (Table 10) [25].
The important signaling pathway modulated by tocotrienols is "Eukaryotic translation Initiation Factors" (EIF2) signaling pathway at the top of the list (Table 10). This is involved in protein synthesis and requires many polypeptides. EIF2 is a GTP-binding protein, which initiates specific forms of met-tRNA onto the ribosome. Its important function is to deliver charged initiator met-tRNA to the ribosome, it also identifies the translational starting site [16,25]. Autophagy is a basic catabolic mechanism that involves cellular degradation of unnecessary or dysfunctional cellular components through the actions of liposome ( Figure 9A) [26,27]. Autophagy is generally activated by condition of nutrient deprivation but has also been associated with physiological as well as pathological processes such as development, differentiation, neurodegenerative diseases, stress, infection, and cancer [27][28][29]. The mammalian Target of Rapamycin (mTOR) kinase is a critical regulator of autophagy induction, with activated mTOR (AKT and MAPK signaling) suppressing autophagy, and negative regulation of mTOR (AMPK and p53 signaling) promoting it [28]. The autophagy pathway ( Figure 9A) highlights the key molecular events involved in triggering autophagy. Inhibiting the proteasome activity also causes the onset of autophagy, as observed with tocotrienol treatment. Apoptosis is a coordinated energy-dependent process that involves the activation of a group of cysteine proteases called caspases and a cascade of events that link the initiating stimuli to programmed cell death [29]. The two main pathways of apoptosis are the intrinsic and extrinsic pathways. Each pathway requires specific triggers to initiate a cascade of molecular events that converge at the stage of caspase-3 activation ( Figure 9B) [30]. The activation of caspase-3 in turn triggers an execution pathway resulting in characteristic cytomorphological features including cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentation [30]. Further details of intrinsic and extrinsic pathways were found in the attached Ingenuity Apoptosis Signaling Pathway ( Figure 9B), which highlights the key molecular events involved in trigging apoptosis. These are followed by protein ubiquitination, Toll-Like Receptor signaling (TLRs), Signal Transducers and Activators of Transcription (STATs), nuclear factor kappa B (NF-κB) transcription factors pathways play major roles in a variety of cellular processes, such as cell cycle, cell proliferation, apoptosis, DNA repair, transcriptional regulation, cell surface receptors, ion channels regulation have been discussed in several publications [31,32]. These results are consistent with these conclusions and δ-tocotrienol treatment of hepatitis C patients, acts by increasing cell death, and necrosis of malignant tumors, and by decreasing viral infection, cellular growth, and proliferation, Qureshi Page 7 Ann Clin Case Rep. Author manuscript; available in PMC 2022 December 19.
decreasing endocrine system disorders such as diabetes mellitus, and mobilization of calcium. Therefore, tocotrienols can safely be used for hepatitis C patients, without any side effects. This is first report describing RNA-sequence analysis of δ-tocotrienol treated plasma total mRNAs obtained from chronic hepatitis C patients that acts via multiple-signaling pathways without any side-effect. These studies may lead to development of novel classes of drugs for the treatment of chronic hepatitis C patients [25]. Diabetes mellitus is a metabolic disorder identified by hyperglycemia due to insulin resistance. Impaired serum/plasma fasting glucose, HbA1c, hs-CRP are biomarkers, normally used to determine onset of diabetes. δ-Tocotrienol, vitamin D 3 and resveratrol (nutritional supplement-NS-3) are potent anti-cholesterolemic, anti-oxidative and anti-inflammatory agents. It was hypothesized that a mixture of δ-tocotrienol, vitamin D 3 resveratrol (NS-3; Figure 5) will be more effective treatment for reducing diabetes biomarkers as compared to its individual components in people with type 2 Diabetes Mellitus (T2DM) [33]. Therefore, estimations of NS-3 mixture and its individual components were carried out to test the hypothesis, on diabetes and inflammatory biomarkers, using  (Table 11) [34]. Treatment with individual components showed significant decreases but were less effective than the mixture (Table 12) [34]. The mixture and its components did not induce autophagy in these PBMC ( Figure 10)  (Table 13A, 13B) [35]. Gene expression of up-regulated miR-29b-3p modulates (GLUT4, insulin resistance), miR-624-5p (nephropathy biomarker), miR-361-5p (chronic inflammation), miR-130a-3p (glucose metabolism, insulin secretion), miR-3912-3p (lipid metabolism), and miR-11401 (cellular transcription). The miR-374c-5p (insulin resistance), miR-4326 (HbA1c level)), miR-874-3p (β-cell function) are down-regulated of NS-3 treated people with T2DM (Table 13A, 13B) [35]. Whereas gene expression of molecular functions of messengerRNAs (mRNAs), 42 are up-regulated, out of which mainly associated with ML-1621513 (oxidative/stress), mR-CTD-2349P217 (insulin-mediated glucose-uptake) are up-regulated and mR-CTC-246B1810 (β-cell/biology) ( Table 14A). The 17 down-regulated gene expression of HBB functions as theranostic molecule, also as a hemoglobin glycation in people with T2DM, CTC-246B1810 is involved with several cytokines and β-cell biology in T2DM (Table 14B) [35]. The other important gene AGBL5-IT1 is associated CRISPRclones for T2DM. The RN7SL698P gene expression plays role in many inflammatory T2DM cytokines and its complication in diabetes, and COX5BP7 modulate proper glycemic control in T2DM after NS-3 treatment (Table 14B) [35].
The molecules functions of paired mRNAs-miRNA are found fold changes in gene expression of up-regulated (38) with log ratios of 10.2-1.0 and down-regulated (4) with log ratios of −1.1-1.3 out of a total 1000 genes. The summary of paired mRNAs-miRNAs IPA analyses is described in 54 categories associated with diabetes (Table 15A, 15B). The functions of first ten genes are up-regulated (ZNF525, ZNF28, GNG10, NDUFB4, ORMDL1, S100B, BCKDHA, OXA1L, SBF1, RSU1) and four down-regulated (SET, RAB31, BRD4, KANK2) of paired mRNAs-miRNAs of molecular functions are also discussed further (Table 15A,  post-treatment groups [35]. Furthermore, all these results are supported by their heat map of miRNAs, in which up-regulated gene expression of pre-treatment were down-regulated after post-treatment as shown in Figure 11, whereas summary of various genomic functions of mRNAs of pre-treatment vs. past-treatment were up-regulated two-told to three-fold of people with T2DM [35]. These results collectively identified 92 mRNAs that are upregulated with negative correlation of 14 miRNAs (miR-3074-5p, miR-5481, miR-125a-5p, miR-374c-3p, miR-548-3p, miR-576-3p, miR-1292-5p, miR-296-5p, miR-1304-3p, miR-374-3p, miR-4326, miR-6513-3p, miR-5695, miR-4646-3p), which are down-regulated of post-treatment group of T2DM ( Figure 12). It is clear from this Figure 12 that a single miRNA can regulate multiple targets of mRNAs, for example miRNA-5481 targets several mRNAs associated with T2DM ( Figure 12) [35]. The interaction network of miR-29b-3p is generated using genes/molecules/pathways based on experimentally observed evidence of directly interacting with miR-29b-3p in people with T2DM. The molecules are organized according to their subcellular locations such as extracellular space, plasma membrane, cytoplasm, nucleus, or "other" category ( Figure 13) [35]. The transcriptome expression data of network indicates red shades denote intensities of up-regulation, whereas green shades denote intensities of down-regulation of genes, and gray denote no change in post-treatment group compared with pre-treatment group. For example, LOXL2 enzyme coding gene has log2FC of −2.38 and has a darker green shade as compared to LAMC1 which has log2FC of only −0.19 and hence has lighter shade of green. Whereas the location of TUG1 is specified in "other" category ( Figure 13). There were eighteen (

Conclusions
These results confirm that consumption of δ-tocotrienol plus AHA Step-1 diet causes significant reduction in serum lipid parameters and several cytokines (TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10) at a lower optimum dose of 250 mg/d. The capacity of δ-tocotrienol to modulate inflammation is partly attributable to dose-dependent properties of inhibition/ activation, which may play a major role in future treatment of cardiovascular diseases. The effect of δ-tocotrienol on pharmacokinetics and bioavailability of all eight isomers of tocol indicated that when tocotrienols are supplemented in absence of tocopherols, δ-tocotrienol has better bioavailability, and δ-tocotrienol is converted stepwise to other tocotrienols/ tocopherols. These results also support that tocotrienol, particularly δ-tocotrienol, as a dietary supplement might be useful in the prevention of age-related and chronic ailments. The pharmacokinetics of higher doses of 750 mg and 1000 mg of δ-tocotrienol have confirmed that T max was 3 h to 4 h for all tocol isomers except α-tocopherol (6 h), and these higher doses of tocotrienols are found to be safe and might be useful for the treatments of various types of cancer, diabetes, and Alzheimer's disease. The present results have provided two sets of compounds, anti-inflammatory (for the control of diabetes and cardiovascular disease), and pro-inflammatory for the treatment of cancer and other diseases. These results also demonstrate effectiveness of several natural-occurring compounds with anti-proliferative properties against cancer cells of several organs of humans. Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol and quercetin are very effective for apoptosis of cancer cells in liver, pancreas, prostate, breast, lung, melanoma, B-lymphocytes, and T-cells. The results have provided an opportunity to test these compounds either individually or in combination as dietary supplements in humans for treatment of various types of cancers. The results of fold-change expression data analyzed by "Ingenuity Pathway Analysis" describe the effect of δ-tocotrienol in chronic hepatitis C patients on biological mechanisms at molecular level. It also revealed an insight of correlation of signaling pathways and transcriptional factors. The collective results indicated that tocotrienols inhibit cancer cell proliferation, promotes cell cycle arrest, decreases angiogenesis and acts via multiple signaling pathways. These results clearly indicates that δ-tocotrienol treatment of hepatitis C patients, acts by increasing cell death, and necrosis of malignant tumors, and by decreasing viral infection, cellular growth, and proliferation, decreasing endocrine system disorders such as diabetes mellitus, and mobilization of calcium. Therefore, tocotrienols can safely be used for hepatitis C patients, without any side effects. These results of in vitro and in vivo studies support our hypothesis that NS-3 mixture is more effective in lowering serum levels of several diabetes and inflammatory biomarkers including gene expression markers compared to its individual components in people with T2DM.  (2)    The single dose of 125 mg, 250 mg, or 500 mg of δ-tocotrienol was administered in one day to well-fed healthy subject (11/dose). The blood samples were collected in Ethylene Diamine Tetra Acetic acid (EDTA) glazed tubes at pre-dose (0 h) to post-dose 1, 2, 3, 4, 6, 8, 10 h intervals of each subject. The plasma samples were harvested and processed to carry out normal phase HPLC analyses of each subject as described in [14]. Values are means ± standard deviation (n=11/dose). Values are significantly different at P<0.001 from each other.  Thioglycolate-elicited peritoneal macrophages were prepared from 8-week-old female C57BL/6 (Wild Type), and PPAR-α knockout mice as described previously [23]. The macrophages of each mouse were treated with same 14 compounds as in Figure 6A. The TNF-α was assayed by using ELISA assay kit or assayed for production of nitric oxide by measuring the amount of nitrite using Griess reagent. Data are average of triplicate analyses of each sample as ± SD (standard deviation). Percentage values of each treatment compared to control are at the top of the column. Values in a column not sharing a common asterisk are significantly different at *P < 0.05; **P < 0.01; ***P < 0.001 [19]. The autophagy modulated by δ-tocotrienol treatment of hepatitis C patients: Autophagy is a general term for the basic catabolic mechanism that involves cellular degradation of unnecessary or dysfunctional cellular components through the actions of lysosome. Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with physiological as well as pathological processes such as development, differentiation, neurodegenerative diseases, stress, infection, and cancer. The mammalian target of rapamycin (mTOR) kinase is a critical regulator of autophagy induction [25].    The interaction network of miR-29b-3p was generated using genes/molecules/pathways based on experimentally observed evidence of directly interacting with miR-29b-3p in people with T2DM. There were eighteen (18)   The network images showed mRNA (9), miRNA (10) and 29 overlap of paired mRNA-miRNA (29). The most specific network relevant to present study were from mRNA category (RNA-trafficking, cell-mediated-immune-response, inflammatory-disease, lipid metabolism) and from miRNA (immunological disease, immune-cell-trafficking, hematological-disease) as reported in Table 16B    Effects of δ-tocotrienol (250 mg/d) + AHA Step-1 diet on various cytokines in hypercholestrolemic subjects.    Pharmacokinetic parameters after feeding single dose of various concentrations of δ-tocotrienol (125 or 250 or 500 mg) in one day.         Plasma miRNAs of δ-tocotrienol at 0 h to 3 h (125 mg) and 0 h to 6 h (500 mg) of pharmacokinetic study in humans.  Estimation of plasma tocols by normal phase HPLC of pharmacokinetic human study after feeding single dose of 750 mg of δ-tocotrienol in one day.
Normal Phase-Silica column.    Evaluation of following compounds on several inflammatory biomarkers in PPAR-α knockout mice.   Impact of effective dose of various compounds in different cancer cell lines.   The IC 50 values of various compounds in different cancer cell lines.   Effect of δ-tocotrienol on up-regulation of fold change gene expression of "Molecules" (953) section of IPA analysis in hepatitis C patients.     Summary of impact of placebo supplement or a mixture of (NS-3) or its components after treatment for 24-weeks on various biomarkers of diabetes in serum of people with T2DM.